Septic shock from bacterial endotoxins, triggered by release of lipopolysaccharide (LPS) molecules from the outer wall of gram-negative bacteria, is a major cause of human death for which there has previously been no effective treatment once the complex inflammatory pathways have been activated. Delivery of LPS from external fluids to the cell membrane, and ultimately to the LPS receptors is complex, involving a number of external proteins and other factors (Erridge et al., Microbes Infect. 4:837-851 (2002); Bosshart et al., FEBS Lett. 553:135-140 (2003); Thomas et al., Biochem. Biophys. Res. Commun. 307:133-138 (2003); Rustici et al., (1993) Science 259, 361-365 (1993)). A critical factor in LPS toxicity is the aggregation-state of this amphipathic molecule, e.g., when LPS are packed into lamellar phases toxicity is low, but high when present in non-lamellar phases (Brandenburg et al. Carbohydr. Res. 338:2477-2489 (2003)). As a result, strategies to counteract LPS in serum or cerebrospinal fluid have involved several types of small molecules and proteins, including anti-LPS antibodies, such as transferrin, alpha 2-macroglobulin, Gc-globulin (Berger et al., Clin. Chim. Acta 163:289-299 (1987); Berer et al., Eur. J. Clin. Invest. 20:66-71 (1990)) and MD-2 (Shimazu et al., J. Exp. Med. 189:1777-1782 (1999)).
Lipoprotein particles, such as LPS-binding protein (LBP) are also ligands for LPS (Wright et al., J. Exp. Med. 170:1231-1241 (1989)), in which case a rise in plasma lipid levels during sepsis may be a protective response since LPS bound to other molecules appears to lose its stimulatory effect on cells (Casas et al., J. Surg. Res. 59:544-552 (1995); Bannerman et al., J. Dairy Sci. 86:3128-3137 (2003)), and promotes cytokine tolerance in hepatocytes (Harris et al., J. Endotoxin Res. 9:45-50 (2003)). Both LBP and CD14 promote partitioning of LPS into lipoprotein complexes (Vreugdenhil et al., J. Immunol. 170:1399-1405 (2003); Wurfel et al., J. Exp. Med. 181:1743-1754 (1995)). More importantly, low concentrations of LBP have been shown to enhance the LPS-induced activation of mononuclear cells, whereas increased LBP concentrations inhibit LPS-induced cell stimulation (Gutsmann et al., Infect. Immun. 69:6942-6950 (2001)). LBP also delivers LPS to its receptors at the cell membrane. This dual effect of the protein on LPS function reinforces the importance of the lipid aggregation-state, and confirms that toxicity does not result from mass concentration alone. Related to this effect may be the finding that low doses of antimicrobial agents can lead to exacerbation of the toxicity associated with LPS (Tauber et al., J. Infect. Dis. 156:456-462 (1987); Mertsola et al., Pediatr. Infect. Dis. J. 8:904-906 (1986)).
Lipoteichoic acid (LTA) is a surface-associated adhesion amphiphile found in the cell walls of most gram-positive bacteria, and acts as a regulator of autolytic wall enzymes (muramidases). The toxin is released from the gram-positive bacterial cells, mainly after bacteriolysis induced by lysozyme, cationic peptides from leucocytes, or beta-lactam antibiotics or leukocytic mediators, and plays an important role both in the colonization of bacteria and the consequent release of cytotoxic mediators in colonized organs. LTA binds non-specifically to membrane phospholipids in target cells, or it binds specifically to CD14 and to Toll-like receptors (TLRs). When LTA is bound to a target, it can interact with circulating antibodies and activate the complement cascade to induce a passive immune kill phenomenon (Hummell et al., J. Clin. Invest. 77(5):1533-1538 (1986)), and it triggers numerous events associated with sepsis, including the respiratory burst and the release by neutrophils and macrophages of reactive oxygen and nitrogen species, acid hydrolases, arachidonic acid metabolites, highly cationic proteinases, bactericidal cationic peptides, growth factors, stimulators of chemotaxis and phagocytosis, and cytotoxic cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), inflammatory chemokines, such as IL-8, and platelet-activating factor (PAF). The cytokines then bind to cytokine receptors on target cells and initiate inflammation, as well as activating both the complement pathways and the coagulation pathway, in a manner similar to endotoxin (LPS) from gram-negative cell walls, and in fact, may act in synergy to amplify cell damage. Consequently, innate immune defenses, such as the inflammatory response, the complement pathways, and the coagulation pathway become harmful to the body when there is an excessive production of cytokines. LTA also inhibits platelet aggregation, which may contribute in part to bleeding diathesis, which is a characteristic response in gram-positive septicemic patients. Thus, LTA shares many pathogenetic properties with LPS endotoxins.
In animal studies, LTA has been reported to induce arthritis, nephritis, uveitis, encephalomyelitis, meningeal inflammation, and periodontal lesions, and also to trigger cascades resulting in septic shock and multi-organ failure. However, LTA binding to targets can be inhibited by antibodies, phospholipids, and specific antibodies to CD14 and TLR. In vitro, the release of LTA can be inhibited by non-bacteriolytic antibiotics, and by polysulphates, such as heparin, that apparently interfere with the activation of autolysis.
Thus, evidence has shown that LTA is a significant virulence factor in infectious and post-infectious sequelae caused by gram-positive bacteria, making the development of effective anti-bacteriolytic drugs and multi-drug strategies to attenuate LTA-induced secretion of pro-inflammatory agonists of great importance to combat septic shock and multi-organ failure. Moreover, gram-positive and gram-negative infections often co-exist, producing a synergistic effect of LPS and LTA, and results in a clinical septicemia of greater severity than infection by either pathogen alone. Thus, LTA sits at the heart of septicemia and blockade of its binding could confer significant clinical activity.
Naturally occurring amphiphilic lysosphingolipid molecules control vital functions of the cell through their interaction with specific receptors to modulate a variety of signaling pathways. Proliferation, differentiation and programmed death result from a fine balance of signals, among which sphingosine and structurally related molecules play fundamental roles, acting as either first or second messengers. Sphingosylphosphocholine (SPC) is a deacylated derivative of sphingomyelin, which is known to accumulate in Niemann-Pick disease type A.
Sphingosine-1-phosphate (S1P) is an important cellular metabolite, derived from ceramide that is synthesized de novo or as part of the sphingomyelin cycle (in animal cells). In plasma, it can reach a concentration of 0.2 to 0.9 μM, and is found in association with the lipoproteins, especially the HDL (Goetzl et al, J. Biol. Chem. 275(19):14573-14578 (2000)). It has also been found in insects, yeasts and plants.
S1P is a highly active cellular growth factors that is generated enzymatically from cellular membrane precursors and secreted by activated platelets, leukocytes, epithelial cells, and some types of tumors. S1P is stored in relatively high concentrations in human platelets, which lack the enzymes responsible for its catabolism, and it is released into the blood stream upon activation by physiological stimuli, such as growth factors, cytokines, and receptor agonists and antigens. It may have a critical role in platelet aggregation and thrombosis and could aggravate cardiovascular disease. On the other hand, the relatively high concentration of the metabolite in high-density lipoproteins (HDL) may have beneficial implications for atherogenesis. For example, reportedly S1P together with other lysolipids, such as SPC and lysosulfatide, may be responsible for the beneficial clinical effects of HDL by stimulating the production of the potent antiatherogenic signaling molecule nitric oxide by the vascular endothelium. In addition, S1P functions intracellularly to regulate calcium mobilization and cell growth in response to a variety of extracellular stimuli. However, Yamamura et al., FEBS Lett. 382:193-197 (1996) dismissed the speculation that S1P could physically interact with actin-binding proteins, demonstrating that S1P was poorly active and yielded only a maximal dissociation of 10%.
Gelsolin is a normal serum protein, but unlike other mammalian proteins, gelsolin has both cytoplasmic and secreted isoforms derived by alternative slicing of the message from a single gene (Sun et al., J. Biol. Chem. 274:33179-33182 (1999)). Although originally identified as a cellular protein, cytoplasmic gelsolin also exists as an abundant secreted isoform of nearly identical structure and is a product of the same gene (Kwiatkowski et al., Nature 323:455-458 (1986)). It has a six-fold sequence repeat structure that is highly conserved among gelsolins of vertebrate species, and that is characteristic of a large family of gelsolin-related proteins (Kwiatkowski, Curr. Opin. Cell Biol. 11:103-108 (1999)).
Secretory gelsolin, now called “plasma gelsolin,” circulates in human and rodent blood at concentrations of 250±50 μg/L (Lind et al., Am. Rev. Respir. Dis. 138:429-434 (1988)). Plasma gelsolin has a processed signal sequence, but is otherwise identical to the cellular form, with the exception of a 23 amino acid stretch at the amino terminus of the molecule, designated the “plasma extension.” Therefore, the plasma form of gelsolin is slightly larger (84 kDa), than the cellular variant (82 kDa). Recombinant human gelsolin (rhGSN) (Biogen IDEC, Inc., Cambridge, Mass.) is produced in E. coli, and though it has the same primary structure as native protein, under standard conditions of purification, it differs from natural human plasma gelsolin by a disulfide bond that is present in the natural protein. The recombinant protein is, therefore, properly oxidized after purification, and its structure and functions are indistinguishable from purified human plasma gelsolin (see, Wen et al., Biochemistry 35:9700-9709 (1996)).
Although secreted by many tissues, the major source of human plasma gelsolin is straited muscle (Kwiatkowski et al., J. Biol. Chem. 263:8239-8243 (1988)). This form of gelsolin appears to be distributed throughout extracellular fluids and has a typical residence time in plasma (Lind et al., J. Clin. Invest. 78:736-742 (1986)). It is not modified post-translationally by glycosylation or other reactions, nor is it an acute phase reactant. In fact, little information exists concerning its function or regulation of its production. For example, gelsolin did not bind S1P, nor did it deliver S1P to rat cardiac myocytes (Goetzl et al., supra, 2000). However, it is known that levels of gelsolin drop in clinical settings when inflammation or sepsis occurs, i.e., tissue injury associated with breakdown of membrane barriers and exposure of actin to the extracellular environment results in reductions in plasma gelsolin levels (Lee et al., N. Engl J. Med326:1335-1341 (1992)).
As a result, plasma gelsolin depletion precedes and predicts complications of severe injury, such as respiratory failure and death in animals including humans (Mounzer et al., Am J. Respir. Crit. Care Med. 160:1673-1681 (1999); DiNubile et al., Blood 100:4367-4371 (2002)). For example, a drop in plasma gelsolin levels to <50% normally is a strong predictor of adverse clinical outcomes associated with massive inflammation, although neither a causative role, nor a treatment has been identified based on this finding (Dahl et al., Shock 12:102-104 (1999); Dahl et al., Crit. Care Med. 31:152-156 (2003); Lind et al., 1988, supra; Smith et al., Blood 72:214-218 (1988); Suhler et al., Crit. Care Med. 25:594-598 (1997)). However, replacement of depleted plasma gelsolin levels has attenuated the pathologic sequelae of severe primary injuries in animal models (Christofidou-Solomidou et al., J. Investig. Med. 50: 54-60 (2002); Rothenbach et al., J. Appl. Physiol. 96:25-31 (January 2004)), supporting the findings of the present invention.
In response to acute trauma and/or infection, abundant but normally intracellular G-actin (monomeric actin) is released into extracellular spaces from damaged or dying cells and tissues and circulates in fluid and blood. Once released, G-actin has a strong tendency to polymerize to F-actin. The persistance of filaments of F-actin in the microvasculature of mammals can result in venous obstruction, pulmonary microthrombii and/or endothelial injury, and induces or enhances platelet agglutination in the blood, thereby triggering thrombus development (Scarborough et al., Biochem. Biophys. Res. Commun. 100:1314-1319 (1981)). Combined, these effects alter the characteristics of normal vascular flow in mammals, and in turn, can result in actin toxicity disorders and contribute to the pathogenesis of organ injury at sites removed from the primary insult (Dahl et al., Crit. Care Med. 26:285-289 (1998); Lee et al., 1992, supra; Mounzer et al., 1999, supra, see also U.S. Pat. No. 5,593,964).
However, various actin-regulating proteins contribute to the reversible conversion of filaments (“gel”) and monomers (liquid “sol”), and changes occur depending on extracellular stimuli (see, U.S. Pat. No. 6,271,353). Plasma gelsolin and secreted Gc-globulin act in a coordinated manner, representing an “actin-scavenger system,” to depolymerize and remove the actin filaments released from damaged cells. Gelsolin binds to both monomeric and filamentous actin (Yin et al., Nature 281:583-586 (1979); Yin et al., NJ. Biol. Chem. 259:5271-5276 (1984)), although following injury, gelsolin preferably binds to and severs the actin filaments to promote rapid depolymerization (Sun et al, 1999, supra), whereas Gc-globulin binds to the actin monomers to shift the actin monomer/polymer equilibrium back toward depolymerizations and prevent repolymerization (Goldschmidt-Clermont et al., J. Clin. Invest. 81:1519-1527 (1988)).
This binding requires the presence of micromolar concentrations of calcium (Ca2+), which may include added calcium or endogenously available Ca2+ in the patient. Moreover the binding is very tight, having a dissociation constant in the nanomolar range. In fact, when gelsolin binds actin filaments in the presence of calcium, it ruptures or severs the filaments at the binding site by breaking the noncovalent bonds holding actin monomers together within the polymer (Janmey et al., Biochemistry 24:3714-3723 (1985)). Following the actin severing reaction, gelsolin remains tightly bound to one end of the polarized actin filament, the end conventionally defined as “barbed.” This is also the end that rapidly exchanges monomers.
Removing calcium by chelation does not dissociate gelsolin from the barbed ends of the actin filaments. Instead, phosphoinositides (also referred to as phosphorylated inositol phospholipids, or “PPIs”), which are important signal transduction intermediates, effect this separation at the plasma membrane. Gelsolin binds with high affinity and selectivity to PPI and to lysophosphatidic acid (LPA) (Janmey et al., J. Biol. Chem. 262:12228-12236 (1987); Meerschaert et al., Embo J. 17:5923-32 (1998)). PPIs, therefore, regulate the intracellular actin-binding function of gelsolin, leading to the hypothesis that a reciprocal relationship, between calcium transients and membrane phosphoinositide synthesis and degradation, regulates gelsolin and cellular actin remodeling responses (Stossel, J. Biol. Chem. 264, 18261-18264 (1989)). Gelsolin binding to LPA, modulates its receptor-mediated biological effects, leading to the belief that it may act as a carrier of LPA to some cellular receptors, and buffers bioactive inflammatory lipid mediators (Goetzl et al., supra, 2000).
Calcium and phosphoinositides also control the actin-binding functions of plasma gelsolin in vitro (Janmey et al., Chem. Biol. 5:R81-85 (1998)). The PPI regulatory site of gelsolin resides within a 20 residue linear sequence that connects the first and second folded domains of the protein. Biochemical and mutational studies have implicated 10 strategically organized basic and hydrophobic amino acids (160-169, SEQ ID No:1 QRLFQVKGRR) in the 684-residue plasma gelsolin molecule that accommodate tight binding to the negatively-charged phosphomonoesters and hydrophobic acyl chains of anionic phospholipids (Janmey et al., J. Biol. Chem. 267:11818-11823 (1992). Synthetic peptides of this sequence have a PPI binding affinity similar to that of intact gelsolin (Id).
The methods used to measure the function of gelsolin, have exploited gelsolin's calcium-dependent actin filament severing function, or actin monomer nucleation activities, or the actual mass of gelsolin, using western blotting or immunochemical (usually ELISA) assays. The assays used to quantify gelsolin have relative advantages and disadvantages. For example, the assays measure only severing activity that is directly measurable by fluorimetry (Janmey et al., Blood 70:524-530 (1987)) and the assays are specific for free gelsolin, but actin and lipid binding to gelsolin inhibit this function. Nucleation activity and the structural assays do not differentiate free from complexed gelsolin. However, different assays have thus far yielded very similar results for plasma gelsolin levels in control samples, and generally consistent degrees of diminution in plasmas from injured animals or humans, with the exception that severing activity is lower than expected. However, there are also unexplained data from studies showing diminished severing, but not total gelsolin activity in serum samples with no evidence of actin, suggesting that another ligand may have bound the gelsolin.
While there is no apparent relation between the function or metabolism of LPS and either LPA or PIP2 (phosphatidylinositol-4,5-bisphosphate), these lipids share two unusual and essential structural characteristics, and as acidic lipids, they superficially resemble LPS in that all have phosphomonoesters juxtaposed to a hydrophobic interface (Erridge et al., 2002, supra). The active site of each molecule contains a phosphomonoester emanating from a carbohydrate base, glycerol in the case of LPA, and a sugar ring for LPS and PIP2. These moieties, which are present in few other lipids, present possibilities for electrostatic and hydrogen-binding interactions that stabilize both lipid-protein and lipid-lipid complexes ((Liepina et al., Biopolymers 71:49-70 (2003)). Also, none of these lipids forms lamellar phases by itself (e.g., Flanagan et al., Biophys. J. 73:1440-1447 (1997)), LPA and PIP2 binding to gelsolin appears to be strongest when the lipid is in micelles or putative lipid clusters within bilayer vesicles (Meerschaert et al., Embo J. 17:5923-5932 (1998); Tuominen et al., Eur. J. Biochem. 263:85-92 (1999); Janmey et al., J. Biol. Chem. 262:12228-12236 (1987)).
Plasma gelsolin depletion contributes to the pathophysiology of pulmonary microvascular dysfunction resulting from a primary burn- or bacterially-induced inflammation, and inflammation-induced lung injury (Rothenbach et al., 2004, supra). A blatant clinical example of secondary tissue injury associated with, and resulting from, a depletion of gelsolin, is adult respiratory distress—multiple organ dysfunction syndrome (ARDS/MODS) (Ware et al., N. Engl. J Med. 342:1334 (2000)). Plasma gelsolin levels have dropped to reportedly, on average, 30% of normal values in established ARDS cases (Yin et al, 1979, supra). An unimpressive track record of pharmacologic interventions in established ARDS, suggests that once full-blown ARDS occurs, it may no longer be possible to inhibit the broad spectrum of activated inflammatory mediators that are involved.
Accordingly, at the present time, there is no agent to clinically neutralize endotoxins, or prevent or avert the resulting bacterial sepsis, and often death. A need has remained, therefore, until the present invention, to better understand PPI-binding proteins and the lipid ligands of these proteins, how they affect the function of gelsolin, how altering PPI levels in the body works to reorganize the actin cytoskeleton, and the role of these lipids and lipid binding proteins, such as gelsolin, in inflammation, particularly with regard to developing methods for treating, alleviating, or even preventing, the onset and pathology of bacterially-caused endotoxemia, sepsis, inflammation-induced pulmonary microvascular dysfunction following severe burns or myocardial infarction, ARDS or cystic fibrosis.